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Keywords:

Background

Heart failure places an increasingly heavy disease burden on populations world-wide,
leading to a need to understand basic mechanisms underlying the disease [1]. Identification of basic mechanisms that promote the downward cascade of heart failure
holds the promise to develop targeted new therapeutic strategies. Altered ion homeostasis
contributes to hypertrophic heart growth, which impairs the heart’s ability to pump
effectively and commonly progresses to heart failure. Cardiac Na+/H+ exchanger 1 (NHE1) is central to maintenance of intracellular pH. Indeed, experimental
and clinical studies demonstrated the pathophysiological implications of increased
NHE1 activity during an ischemic episode and in hypertrophy [2-4]. NHE1 activity is detrimental to the myocardium as a result of increased intracellular
Na+ load, leading to elevated intracellular Ca2+ through the action of Na+/Ca2+ exchanger, NCX1. In some studies, increased cardiac expression of NHE1 protein appears
to be involved in the subsequent pathological changes [5]. In human heart failure, however, enhanced NHE1 activity is not correlated with increased
NHE1 expression, suggesting a role for activation by post-translational mechanisms
[6].

Sustained NHE1 activity requires an acidifying pathway, such as Cl-/HCO3- exchange mediated by AE3, since NHE activity alkalinizes the cell, resulting in self
inactivation through a cytosolic modifier site [7,8]. Hyperactivation of NHE1 and AE3 exchanger are associated with hypertrophic heart
growth, in a model of spontaneously hypertensive rats [9]. Maximal activity of AE3 and NHE1 require the catalytic activity of the enzyme carbonic
anhydrase (CA), which provides the HCO3- and H+ substrate for the two transporters [10-12]. Treatment of cultured rat cardiomyocytes with the CA inhibitor, ethoxyzolamide (Cardrase),
prevented hormonally-induced hypertrophy and reversed it once established [13]. ETZ also normalized spontaneous Ca++ transients induced by pro-hypertrophic hormones, indicating that CA has a role in
the elevated Ca++ found in the hypertrophic heart [13]. The identity of the CA isoform responsible for the anti-hypertrophic effects was
not established in the earlier work. Together, CA inhibition, a therapy previously
used for diuresis targeting hypertension and heart failure [14], may be an effective therapeutic approach towards mitigation of the heart disease.

Beneficial effects of NHE inhibition in the failing heart have been suggested on the
basis of cellular signalling mechanisms and experimental studies [5,15]. Here we examined the expression of the CA isoforms, CAII and CAIV, in normal, hypertrophic
and failing human hearts. Our data lead to the conclusion that increased CA expression
is a marker of hypertrophic heart, which progresses towards failure, and suggests
that CA inhibition is a point to intervene in the hypertrophic cascade. Limiting substrate
availability for NHE1 and AE3 intracellular pH regulatory mechanisms by inhibition
of CA will impair the signals that trigger the hypertrophic heart growth.

Methods

Human ethics and heart sample collection

The study was approved the Human Research Ethics Board, Faculty of Medicine and Dentistry,
University of Alberta and all patients gave written informed consent. To avoid possible
disease-specific confounding factors, only samples from patients with early-stage
hypertrophy (aortic stenosis, valve replacement or coronary artery by-pass surgery)
were used.

End-stage failing hearts (severe cardiomyopathy, heart transplantation), were biopsied
following explantation associated with cardiac transplant surgery. Samples were collected
from right or left free ventricular wall as indicated. Endomyocardial biopsy samples
(EMBs), from patients who were referred to the University of Alberta Hospital for
evaluation of cardiomyopathy, were collected by needle biopsy (average diameter 3
mm), and immediately placed into microcentrifuge tubes containing 4°C RNAlater® (Qiagen, Canada) for storage.

RNA isolation and cDNA synthesis

RNA was isolated from human heart ventricles and human brain cortex (from Cooperative
Human Tissue Network (http://www.chtn.nci.nih.gov/webcite)), and used as a control) with RNeasy Micro kit (Qiagen, Canada), according to the
manufacturer’s instructions. Isolated RNA was treated with DNase I (2 U/ng of RNA;
Qiagen) at 22°C, 15 min. RNA integrity was confirmed by agarose gel electrophoresis.

cDNA synthesis was carried out with Superscript III reverse transcriptase (Invitrogen,
Life Technologies), according to the manufacturer’s instructions. cDNA sequences were
obtained from the GenBank sequence database of the National Centre for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/webcite). Primers were designed with the Oligo software of the DNA Star program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgiwebcite). In conventional RT-PCR, all primers generated only one amplification band visualized
by agarose gel electrophoresis. Sequences for all PCR primers are presented in Additional
file 1: Table S1.

Real-time qPCR

Real-time reverse transcription PCR- Real time PCR was performed with Corbett Rotor-Gene
3000 Real Time analyser (Corbett Life Science, Australia). Real time PCR reaction
contained 25 μL with 12.5 μL of 2X Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen),
5 pmol/primer and 5 μL of template and cDNA prepared from 100 ng total RNA. Cycle
threshold values (Ct) were obtained for CAII, CAIV, CAXIV, ANP, BNP, and GAPDH. GAPDH,
assumed not to vary between samples, was used to normalize for differences in the
efficiency of mRNA isolation from the samples as follows. Ct values were corrected
for each sample by addition or subtraction of cycles so that GAPDH Ct values were
the same.

Isolation and culture of cardiomyocytes

Adult mice were anesthetized with sodium pentobarbital, 150 mg/kg i.p. Animal protocols
were approved by the University of Alberta Animal Policy and Welfare Committee and
performed in accordance with Canadian Council on Animal Care guidelines. Hearts were
excised, and ventricular myocytes obtained by enzymatic dissociation [13].

Statistics

Data are expressed as mean ± SEM. Student Paired t-test or one-way ANOVA followed
by Neuwman-Keuls Multiple Comparison post-test analysis, when appropriate, were used
to compare data. P < 0.05 was considered of statistical significance.

Results

Patient cohort

Clinical conditions of two patient cohorts are summarized. Patients undergoing cardiac
interventions had good prognosis and good cardiac contractility measured by left ventricular
ejection fraction values of 59 ± 3% (n = 14, Additional file 2: Table S2). Hearts from patients undergoing cardiac transplantation had poor contractility,
left ventricular ejection fraction values of 20 ± 2%, and subjected to cardiac transplantation
(n = 13, Additional file 3: Table S3). Patients with prevalence of aortic stenosis (Additional file 2: Table S2), presented heart disease with left ventricular remodeling, including mild
to moderate left ventricular concentric hypertrophy, non-hypertrophic right ventricles,
and non-left ventricular dysfunction (Stage B of heart failure). Conversely, patients
with cardiomyopathies (Additional file 3: Table S3) presented ventricular dilation and severely symptomatic heart failure
(Stage D of heart failure). Hearts were classified according to established criteria
[19]. Importantly, there was no significant difference in age between the two groups.
The overall population with aortic stenosis presented at an average age of 61 ± 4
years, with overrepresentation of males (86%), whereas patients with dilated cardiomyopathies
presented at an average age of 51 ± 4 years, and were also overrepresented by males
(69%).

Analysis of gene expression in human heart biopsies

We examined whether the mRNA and protein expression of carbonic anhydrase genes are
altered by cardiac hypertrophy. We analyzed samples derived from single endomyocardial
biopsies (EMB), and ventricular slices, respectively, from 27 human samples. An average
of 1 mg of total RNA, isolated from each EMB, was subjected to reverse transcription
and quantification of transcript abundance by real-time PCR. To verify the specificity
of the real-time RT-PCR data, we performed RT-PCR using the designed pair primers
(Additional file 1: Table S1) and resolved the products on 1% agarose-ethidium bromide gels. A single
band was found for CAII, CAIV, CAXIV, Atrial Natriuretic Peptide (ANP), and Brain
Natriuretic Peptide (BNP) transcripts, using either brain cortex, or human heart ventricle
RNA (Figure 1A). A weak band was detected for BNP in brain cortex, suggesting low expression in
this tissue. The presence of a single band in each case indicates the specificity
of amplification, so that quantitative real-time PCR data reports levels of a single
species.

Real time reverse transcription PCR quantified the abundance of transcripts in the
heart samples. Cycle threshold (Ct) values in real time-PCR were used as a measure
of transcript abundance, where higher threshold values correspond to lower mRNA abundance
and each change of 1 cycle threshold corresponds to a two-fold difference in message
abundance. To correct for differences in total RNA abundance between samples, each
sample was analyzed for ANP, BNP and the CA genes, and for GAPDH (Figure 1B). To correct for variations in the amount of mRNA assayed, cycle threshold values
were corrected by the Ct for GAPDH, assumed to be present at a constant baseline level.

ANP and BNP are fetal genes whose expression is induced during hypertrophic cardiomyocyte
growth, or during heart failure [20,21]. We found marked increases of ANP and BNP expression in all hypertrophic or dilated
ventricles (failing heart), compared to non-hypertrophic non-dilated ventricles (non-failing
hearts), or compared to ventricles with mild/moderate hypertrophy or dilation (non-failing
hearts) (Figure 1B). Remarkably, CAII and CAIV message increased at least two-fold in hypertrophic
ventricles, and 16-fold in failing hearts (4 Ct difference), compared to non-hypertrophic,
non-dilated non-failing ventricles (Figure 1B). CAXIV mRNA expression did not increase in hypertrophic ventricles of non-failing
heart and failing ventricles. We conclude that hypertrophic ventricles express elevated
levels of CAII, CAIV, ANP, and BNP, message, relative to non-hypertrophic non-dilated
ventricles, which increased in failing ventricles, as evaluated by qRT-PCR. Expression
of ANP, BNP, CAII, CAIV, and CAXIV did not differ between right and left failing ventricles
(Figure 1C).

Expression of carbonic anhydrase proteins

To quantify the level of CA protein expression in hearts, immunoblots were performed.
Because of the small size of material collected in EMBs, it was not possible to evaluate
protein expression in samples from patients with good prognosis (Additional file 2: Table S2). Ventricular samples collected from explanted failing explanted hearts
(right or left ventricles), but with no signs of either hypertrophy or dilation, were
compared to hypertrophic or dilated failing ventricles (Additional file 3: Table S3).

CAII, a near-ubiquitous cytosolic isoform, is expressed in mouse embryonic and fetal
heart, and adult mouse heart [13,22]. We examined the expression of CAII in mouse and human hearts, by immunoblotting.
Immunoreactivity was observed in HEK293 transiently transfected with hCAII cDNA, and
endogenously in HEK293 cells transfected with empty vector, (Figure 2A). Human heart ventricular samples from explanted hearts, and cardiomyocytes freshly
isolated from adult mouse heart, showed robust and moderate CAII expression, respectively
(Figure 2A). No immunoreactivity was found on parallel blots incubated with non-immune rabbit
serum (not shown). The presence of endogenous CAII in HEK293 cells has been reported
previously, using the same antibody [10].

To examine the role of CAIV and CAXIV in the failing heart, we studied their expression
(Figure 2B,C). Immunoreactive bands, corresponding to the expression of CAIV and CAXIV, were
observed in HEK293 cells transiently transfected with rabbit CAIV, or mouse CAXIV
(Figure 3A), cDNAs. HEK293 cells transfected with empty vector did not reveal CAIV or CAXIV
immunoreactive bands, indicating specificity of the antibodies. Human failing ventricles
and isolated adult mouse cardiomyocytes, showed modest and significant CAIV and CAXIV
expression, respectively (Figure 2B,C). Specificity of the CAIV antibody is indicated by the lack of band in untransfected
HEK293 cells (Figure 2B) and the specificity of the antibody has been previously assessed [23]. Similarly, in untransfected HEK293 cells there is a faint band at the CAXIV position,
possibly arising from spillover from the much stronger signal in CAXIV-transfected
cells (Figure 2C) and the antibody’s specificity has previously been assessed [18].

Figure 3.Expression of CAII, CAIV, and ANP proteins in adult human ventricle.A, Lysates were prepared from hypertrophic/dilated (H/D), or non-hypertrophic/non-dilated
(NH/ND) adult human ventricles, from explanted failing hearts. Left panels, failing
ventricles with no signs of either hypertrophy or dilation were compared with hypertrophic
or dilated failing ventricles. Protein samples (30 μg) were resolved by SDS-PAGE,
transferred to PVDF membrane, and probed with anti-CAII, anti-CAIV, anti-ANP, and
anti-α-actinin antibodies. Right panels, samples from left (L) and right (R) ventricles
were directly compared. Filled arrow indicates position of protein. B, Summary of the protein expression normalized to α-actinin. Values are expressed
relative to the non-hypertrophic/non-dilated protein expression; (n = 5). C, Summary of the protein expression normalized to α-actinin. Values are expressed
relative to the control right ventricle protein expression; (n = 5). *Indicates statistically
significant difference (P < 0.05).

Some patients with end-stage heart failure presented with either left or right ventricles
with no signs of hypertrophy or dilation (Additional file 3: Table S3). To evaluate whether the CA isoform expression was altered in failing
ventricles with hypertrophic or dilated versus non-hypertrophic non-dilated ventricles, protein expression was quantified by densitometry
of the immunoblots (Figure 3). CAII, CAIV, and ANP proteins could be clearly identified on immunoblots (Figure
3). CAII, CAIV, and ANP protein expression increased ~2 and ~2.5-fold in hypertrophic/dilated
ventricles (Figure 3). CAXIV showed a slight increase, ~35%, in dilated failing ventricles compared to
non-hypertrophic non-dilated failing ventricles, but did not reach statistical significance
difference.

In the failing hearts, CAII, CAIV, and ANP protein expression increased in ventricles
without signs of hypertrophy or dilation compared to hypertrophic or dilated ventricles,
demonstrating that these genes increased under hypertrophic conditions independent
of the contractile performance of the heart. Conversely, failing explanted heart,
showed no differences in CAII, CAIV, CAXIV, and ANP protein expression, in non-hypertrophic
non-dilated right ventricles, compared to non-hypertrophic non-dilated left ventricles
(Figure 3A right panel and 3B, n = 3).

Discussion

This study examined whether altered carbonic anhydrase expression plays a role in
human heart failure. Heart failure ranges in severity from moderate impairment in
cardiac function, to significant damage that leaves the heart unable to manage its
workload [24]. This study demonstrates that CAII and CAIV expression increased in failing human
ventricles. These enzymes need to be considered for their contribution to the progression
of heart failure and as prognostic markers. A caveat to this work is that the extremely
small amount of material in biopsies prevented assessment of changes of carbonic anhydrase
catalytic activity.

Similar to our findings, elevated CAII expression was observed in rats with spontaneous
hypertension and heart failure (SHHF) [25]. Furthermore, mice that develop angiotensin II-induced cardiac hypertrophy (TG1306/1R,
TG), and dilated cardiomyopathy with aging [26], had increased expression of CAII, CAIV, and CAXIV, mRNA, in addition to elevated
mRNA for low-activity secreted CAVI [27], suggesting that induction of carbonic anhydrases is a feature of cardiac hypertrophy.
Others observed no difference in ANP mRNA abundance between left or right ventricle
from control WKY rats, but levels increased in either left or right hypertrophic ventricles
of naturally occurring biventricular hypertrophic rats [28].

In the human heart, the right ventricle has important anatomical and functional differences
from the left ventricle. The right ventricle is a thin-walled, low-pressure structure
that unlike the left ventricle receives most of its blood supply during systole. It
has a complex, crescent shape in contrast to the left ventricle with a simple ellipsoid
form. Differences between the two ventricles are, however, mostly related to their
functions. As the left ventricle must pump blood much further and with more resistance
than the right does, the muscular wall of the left ventricle is far thicker to produce
the necessary force. Differences in the message and protein for CAs, ANP, and BNP
genes expressed in healthy left ventricles compared to healthy right ventricles have
not been evaluated here. We were unable to complete this analysis since we could not
obtain comparable non-diseased ventricular material.

Carbonic anhydrases work with the AE3 Cl-/HCO3- exchanger and NHE1 Na+/H+ exchanger to promote cardiomyocyte hypertrophy, as is found in heart failure (Figure
4). Both AE3 and NHE1 bind to the cytosolic enzyme, CAII, to form a transport metabolon,
the complex of a membrane transport protein and the metabolic enzyme responsible for
metabolism of the transported substrate [10,11,29]. CAII catalytic activity (CO2 + H2O↔ HCO3- + H+) produces HCO3- and H+ for efflux by AE3, and NHE1, respectively (Figure 4). Combined action of AE3 and NHE1 results in net cellular NaCl loading, without change
of pHi, which is consistent with the finding of elevated Na+ and unchanged pHi in pro-hypertrophically-stimulated cardiomyocytes [30,31]. Co-activation of NHE1-CAII and AE3 is pathological as it is self-sustaining and
NHE1 is not subject to inhibition by alkaline pHi, since the co-activated transporters do not change pHi[4]. We previously found that the activity of AE3fl and NHE1 promote hypertrophy and
the hypertrophy-programmed increases expression of the CA enzymes in cultured rat
cardiomyocytes [13]. Sustained NHE1/AE3 activation is itself pro-hypertrophic as elevated Na+ decreases the efficacy of the Na+/Ca2+ exchanger, which normally contributes to maintenance of low cytosolic Ca2+ levels. In turn, sustained elevated Ca2+ is a consummate hypertrophic signal, working through the calcineurin/NFAT signaling
cascade.

Recent studies also point to a pro-hypertrophic role of CAII in rodent hearts [34]. Cardiomyocytes Car2 mice [35], which have a disrupted caii locus, have decreased cardiomyocyte hypertrophy in response to phenylephrine. Moreover,
in rat cardiomyocytes over-expression of a catalytically-null CAII mutant inhibited
cardiomyocyte hypertrophy, in a dominant negative manner.

Here we found that CAII and CAIV mRNA levels rise dramatically in hypertrophied and
failing human hearts. CAIV has also been found to associate with anion exchangers
and to enhance their transport activity, by metabolism of substrate bicarbonate [23]. AE3’s HCO3- efflux activity is maximized by CAIV, as conversion of HCO3- (by CAIV) maximizes the transmembrane [HCO3- gradient, which enhances the rate of HCO3- transport. We propose that AE3, and NHE1, CAII, and CAIV work together to promote
cardiac hypertrophy (Figure 4). Sustained co-activation of AE3 and NHE1 is pro-hypertrophic, and this is exacerbated
by CAII and CAIV, which promote their combined function. The increased expression
of CAII and CAIV expression in hypertrophied human myocardium is consistent with a
pathological feed-forward cascade in which increased CAII/CAIV expression contributes
to hypertrophic signaling, including increased CA expression (Figure 4). To intervene in the hypertrophic cascade present in heart failure we propose that
CAII and CAIV represent targets for anti-hypertrophic therapy. Previously, we found
that the membrane permeant CA inhibitor, 6-ethoxyzolamide (ETZ, Cardrase), which targets
the HTM, intervenes in the feed-forward cascade, preventing and reversing the agonist-induced
cardiomyocyte growth [13].

As a counter-point to this argument, however, the role of carbonic anhydrases in moderating
the global activity of pH regulatory transporters in heart has been suggested to be
modest [36,37]. An alternate explanation for the effects of CAII/CAIV on cardiac hypertrophy could
arise through more direct effects on Ca++ channels. Alterations of cytosolic pH have profound effects on Ca++ channels [38]. Recent findings show that bicarbonate transporters can induce localized changed
of cytosolic pH [39], which could be especially significant in confined microenvironments of cardiomyocytes,
for example T-tubules. Localized changes of pH, arising from carbonic anhydrase catalysis,
could therefore influence Ca++ channel activity, with downstream impact on Ca++-dependent hypertrophic signaling cascade.

The membrane permeant CA inhibitor, acetazolamide (ACTZ, Diamox), blocks the reabsorption
of sodium and potassium by inhibiting CA in the renal tubule [40], and was used as a diuretic in patients with severe congestive heart failure, before
the advent of current loop diuretics like furosemide [41]. Clinically, the status of congestive heart failure in all patients receiving Diamox,
clearly improved [42]. Patients with mild heart failure were adequately controlled with Diamox, whereas
patients with severe heart failure require other diuretics alone, or in combination
with Diamox [42]. More recently, ACTZ was safely used in pediatric patients with heart disease, to
lower serum bicarbonate and acid–base excess, and raise chloride [43]. High-dose diuretic therapy is the primary cause of metabolic alkalosis in pediatric
patients with heart disease, and carbonic anhydrase inhibition improved this condition.

Clinical relevance of the present study includes: 1) Carbonic anhydrase expression
levels, in particular CAII and CAIV, increase during progression of cardiac hypertrophy.
The prognostic value of biomarkers as clinical predictor factors in heart failure
is well-established [44-47] and this work suggests that CAII and CAIV are molecular correlates of hypertrophy.
2) The mainstay of treatment of acute heart failure is diuretic therapy. Diuretics
rapidly improve symptoms associated with volume overload, while there are no data
showing morbidity or mortality increased from the use of chronic diuretic therapy.
CA inhibition with existing drugs could be readily adopted to concomitantly induce
diuresis and inhibit the CA enzymes whose activity increases as hypertrophy escalates.

Conclusion

This study suggests that ventricular hypertrophy/failure and the augmented expression
of the CA in the ventricle may be induced by a common mechanism. Ventricular stretch
arising from an increased ventricular load leads to the induction of CA gene expression.
In this paper we have presented evidence of elevated CA as biomarkers for early detection
of cardiac hypertrophy and heart failure, and proposing a mechanism that could improve
the cardiac performance, by CA inhibition. The ability to distinguish individual patients
at the early stage of heart disease, by detecting elevation of CA expression, might
help to improve their functional status and prevent further circulatory collapse that
will require cardiac transplantation.

Authors’ contributions

Acknowledgements

This work was supported by a grant from the Heart and Stroke Foundation of Alberta.
JRC and BVA are respectively a Scientist of the Alberta Heritage Foundation for Medical
Research and an Established Investigator of the Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET) Argentina. We are grateful to the cardiac surgery
patients and their families for their participation in this study. We thank Patricia
Lo for acting as patient coordinator.

Hunt SA, Abraham WT, Chin MH, Feldman AM, Francis GS, Ganiats TG, Jessup M, Konstam MA, Mancini DM, Michl K, et al.: ACC/AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure
in the Adult: a report of the American College of Cardiology/American Heart Association
Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines
for the Evaluation and Management of Heart Failure): developed in collaboration with
the American College of Chest Physicians and the International Society for Heart and
Lung Transplantation: endorsed by the Heart Rhythm Society.